JP6513133B2 - Microplate reader with controlled gas atmosphere and corresponding method - Google Patents

Description

  This patent application claims the priority of US Provisional Application No. 61/380783 (September 8, 2010), the disclosure of which is incorporated herein in its entirety for any purpose.

  The present invention relates to a microplate reader comprising at least one measuring device and one holding device. At least one measuring device may be configured to transmit light emitted by the sample in the well of the microplate inserted in the microplate reader and / or in the well of the microplate inserted in the microplate reader It is used for the detection of the light influenced by the detected sample. The holding device houses at least one microplate and is used to position the wells of the microplate containing the sample relative to the at least one measuring device. The invention further relates to the individual methods and the special use of such a microplate reader.

  A common microplate reader, known for many years as prior art, is based on the principle of measurement of the luminescence and / or the fluorescence of a sample treated with a reagent. Luminescence or fluorescence generally relates to the emission of light coming from the sample, luminescence being produced by the progress of a chemical reaction in the sample, and fluorescence being produced by the irradiation of excitation light. Such microplate readers are used to observe reactions in samples to which reagents have been added, or to detect specific sample components that become fluorescent.

  It is known that individual microplate readers are equipped with injection devices for adding reagents to the samples in the wells of the microplates inserted and used, respectively, in this microplate reader for the purpose of causing a luminescence reaction. There is. The microplate reader is also known to have an illumination device for illuminating or passing light the sample in the wells of the microplate used in this microplate reader. Such microplate readers are used to measure the fluorescence excited in the sample, or the decrease in transparency (absorbance) produced in the sample.

  The objects of the present invention are microplate readers, individual methods of measuring biological cells in microplate readers, and these allow observation of reactions on the sample and detection of specific sample components under at least approximately physiological conditions. It is to propose the use of a microplate reader.

This object is achieved with the microplate reader as disclosed herein with respect to the first aspect. This microplate reader comprises the following configurations a) to c).
a) At least one measuring device selected from the group comprising a1) to a3) below.
a1) A first measuring device designed to measure the absorbance of a sample in the wells of a microplate used or inserted in a microplate reader.
a2) A second measuring device designed to measure the fluorescence of a sample illuminated with light in the wells of a microplate used or inserted in a microplate reader.
a3) A third measuring device designed to measure the luminescence of a sample in the wells of a microplate used or inserted in a microplate reader.
b) A holding device for receiving at least one microplate and for positioning the sample receiving well of said microplate with respect to at least one measuring device.
c) A control unit for controlling the temperature of the gas atmosphere surrounding the sample receiving well of the microplate used or inserted in this microplate reader.

The microplate reader alternatively further comprises the following configuration d1) or d2).
d1) A separation plate for dividing the inner space of the housing of the microplate reader into an application section and a sample section, comprising at least one opening. The separation plate is designed in the wells of the microplate used in the microplate reader so that light which has been illuminated or influenced by the sample passes from it to the instrument compartment from the sample compartment.
d2) Internal housing located within the housing of the microplate reader. A holding device is disposed therein. The inner housing divides the inner space of the housing of the microplate reader into an instrument section and a sample section surrounding the holding device and comprises at least one opening. It is designed so that light which is illuminated or affected by the sample passes from the sample compartment to the instrument compartment in the wells of a microplate used in a microplate reader.

  The main function of the separation plate and the inner housing is common to these alternative zoning means, ie the sample compartment is affected only when the sample in the sample compartment is needed by the instrument It is to separate from the equipment compartment.

  In the interior space of the housing, the microplate reader can be designed such that the sample compartments are separated from the instrument compartments substantially in a light-tight and substantially airtight manner by means of separating plates and internal housings. The opening may therefore be equipped with a corresponding proofing device. Alternatively or additionally, a blocking device may be provided which extends around the opening. In the described embodiment, the prevention device is mainly to influence the light substantially passing through the opening, ie emitted in the wells of the microplate used in the microplate reader or affected by the sample Light is allowed to pass from the sample compartment to the instrument compartment through the aperture and as much as possible to prevent other light (non-emitted light or light not affected by the sample) from passing through the aperture It is designed.

  A transfer device for mixing and / or circulating the gas present in the sample compartment and / or the gas flowing into the sample compartment may be arranged in the sample compartment. The transfer device may comprise at least one of the following devices: a blower, one or more baffles, a stirring device including one or more paddles, a structured jet nozzle system . Here, the device with one or more baffles may be located near the gas inlet to the sample compartment. A stirrer comprising one or more paddles may be placed near the holding device. The structured jet nozzle system may comprise a plurality of inlet nozzles, which are substantially evenly arranged in the sample compartment, and the gas atmosphere in the sample compartment is displaced upon gas entry through the inlet nozzle. , Will be mixed almost evenly. The inlet nozzles may be arranged to be distributed along the gas inlet line, preferably with equal spacing. The gas inlet lines may each generally run in an arrangement generally parallel to a wall, such as the ceiling or bottom wall of the sample compartment. The arrangement may include a substantially S-shaped arrangement, a plurality of substantially S-shaped arrangements, and / or a substantially spiral arrangement. Alternatively or additionally, the gas inlet line may alternatively or additionally divide or branch into at least a section, one or more sections, two or more gas inlet sublines.

  The control unit may, for example, comprise a built-in computer with corresponding software, which may be connected to or built in the central computer of the microplate reader. Alternatively, the control unit may comprise a computer with corresponding software, which computer is connected with the central computer of the microplate reader and arranged in a separate housing.

  The control unit may be designed to control the composition of a gas atmosphere comprising four, five, six or more different gases. For this purpose, the control unit also comprises a gas sensor for measuring the various gases in the sample compartment, ie for controlling the composition of the gas atmosphere surrounding the sample receiving well of the microplate used in this microplate reader Good. Independently of this, the microplate reader may be provided with a gas inlet, in particular a gas inlet without a gas sensor, which inlet is operable by the control unit to allow gas flow into the sample compartment. is there.

At least one of the various gases is nitrogen (N ₂ ), carbon dioxide (CO ₂ ), oxygen (O ₂ ), carbon monoxide (CO), hydrogen sulfide (H ₂ S), sulfur dioxide (SO ₂ ) It may be selected from the group including

The control unit is preferably an O ₂ sensor for measuring and controlling the oxygen content of the gas atmosphere surrounding the sample receiving well of the microplate used in this microplate reader, and the microplate used in this microplate reader And a CO ₂ sensor for measuring and controlling the carbon dioxide content of the gas atmosphere surrounding the sample well.

  Independently and / or additionally to this, the control unit may be designed to control the humidity and temperature of the gas atmosphere. For this purpose, the control unit may comprise, in the sample compartment, a humidity sensor for measuring the humidity of the gas atmosphere and a temperature sensor for measuring the temperature of the gas atmosphere.

  Independently and / or additionally to this, the control unit may comprise a cooling device for cooling the sample compartment and / or a heating device for heating the sample compartment. Preferably, the heating device is thereby mounted on the separating plate or the inner housing and in heat exchange communication with the gas atmosphere in the sample compartment. A cooling device is mounted at the bottom of the sample compartment and is in heat exchange communication with the gas atmosphere in the sample compartment. The heating device may be mounted on a plate disposed on the sample compartment side of the separation plate or on the inner space side of the inner housing. Preferably, the plate is connected non-thermally to the separation plate and the inner housing. The condensation of humidity and moisture on the microplate and in the wells of the microplate is eliminated by these arrangements of heating and cooling devices. What has been disclosed herein with respect to the cooling device and the heating device also applies to the design of the microplate reader with the separating plate and the inner housing.

  Separate ventilators may be located in the instrument compartment and may be provided in the microplate reader. The ventilator functions to cool the light source (e.g., a lamp) and / or other devices that generate heat. The ventilator may operate and operate independently of the heating device disposed within the sample compartment and the cooling device disposed within the sample compartment.

  In a microplate reader, a first measuring device, a second measuring device and / or a third measuring device respectively measure a light guide having an inlet point for light and an outlet point for light, and light coming out of the outlet point And a light detection device arranged to An access point of light to the first measurement device, an access point of light to the second measurement device, and an access point of light to the third measurement device may be respectively disposed in the sample section.

  Here, the term "light guide" is understood to mean an optical fiber, an optical fiber bundle or a mirror system. A light guide embodied as an optical fiber or fiber optic bundle and a light guide embodied as a mirror system comprises an entrance point for light, an exit point for light and a light guide passage extending from the entrance point to the exit point And along which light can be guided by the light guide.

  In a microplate reader, the individual light guides of the first measuring device, the second measuring device and / or the third measuring device may be embodied as optical fibers or optical fiber bundles. Alternatively, individual light guides can be embodied as a mirror system. A combination of optical fiber and mirror is feasible for illumination of the sample and for detection of light coming from the sample. The light detection device of the first measurement device, the second measurement device and / or the third measurement device may be arranged in the sample compartment or in the instrument compartment.

  In a microplate reader, the first light detection device of the first measurement device and / or the third light detection device of the third measurement device may be arranged in the sample compartment. In a microplate reader, a second light detection device of a second measurement device designed to measure fluorescence in a well may be placed in the instrument compartment.

  The microplate reader may comprise at least one illumination device designed to pass and illuminate the sample in the wells of the microplate used in the microplate reader. The illumination device may comprise a light source and a light guide for guiding at least a portion of the light generated by the light source to the well of the microplate used in the microplate reader and the sample disposed in the well. .

  First, second and / or third lighting devices may be provided for the first, second and / or third measuring devices respectively. However, providing the illumination device in common to the first and second measurement devices, the second and third measurement devices, and the first and third measurement devices, or providing the illumination device in common to the first, second and third measurement devices Is possible.

  The individual light sources of the lighting device may be selected from the group comprising lasers, flash lamps, LEDs (light emitting diodes), laser diodes.

  In particular, in the use of a microplate reader, it may be important to measure and detect the rates of growth and transformation of cell cultures placed as samples in the wells of the microplate. The measurement and detection of velocity can be achieved by measuring and detecting changes in absorbance, fluorescence and / or luminescence of cell culture (sample), preferably changes in luminescence of cell culture (sample) as a function of time .

  In order to measure and detect the speed which travels relatively fast with time, the light source of the lighting device may in particular advantageously need to generate relatively short light pulses. Thus, a pulsed laser, a flash lamp, an LED (light emitting diode) operating in pulsed mode, and / or a laser diode operating in pulsed mode would be suitable light sources for this purpose. A flash lamp not actively cooled may be provided as a light source in the microplate reader. This rush lamp may be located in the equipment compartment. Furthermore, the light generated from this light source may be guided by the light guide to the sample placed in the well of the microplate used in the microplate reader to illuminate and / or pass the light through the sample .

  The microplate reader may comprise a first illumination device designed to pass light to the sample in the wells of the microplate used in the microplate reader, which is arranged in the instrument compartment. Here, the first measuring device may be arranged in the sample compartment. The second measuring device may be arranged in the device compartment. The third measuring device may be arranged in the sample compartment or in the instrument compartment. Here, the first optical access point to the first measurement device, the second optical access point to the second measurement device, and the third optical access point to the third measurement device may be arranged in the sample compartment.

  The microplate reader may comprise a first illumination device designed to pass light through the sample in the wells of the microplate used in the microplate reader. The exit point of the first lighting device may now be arranged in the sample compartment. The microplate reader may further comprise a second illuminator arranged in the instrument compartment and designed to illuminate the sample in the wells of the microplate used in the microplate reader. The exit point of the first lighting device may now be arranged in the sample compartment. The second lighting device may be selected from the group comprising lasers, flash lamps, LEDs (light emitting diodes) and laser diodes.

  The first, second and / or third measuring devices may comprise first, second and / or third optical systems, and at least one of these optical systems and the microplate used (inserted) is Cartesian It is designed to be movable relative to each other in the direction of the Z axis of the coordinate system. The microplate used here (inserted) may be designed to be movable in the direction of the Z-axis. Alternatively or additionally, the first, second and / or third optical systems may be designed to be movable in the direction of the Z-axis. Preferably, the first, second and / or third optical system can be partially lowered into the sample compartment through at least one opening in the separating plate and the wall of the inner housing, eg the ceiling wall Designed.

  In a corresponding method for measuring living cells in a microplate reader (see below) and / or in the use of a microplate reader (see below), for example, in an injection device, at least one well of the microplate Reagents can be added to at least one sample to cause a chemical reaction. When causing a chemical reaction, a change in luminescence properties of the sample, a change in fluorescence and / or a change in absorbance occur with the luminescence reaction. These can be detected, for example, by observing (measuring or detecting) luminescence and / or fluorescence and / or absorbance of a sample, for example, using at least one of measurement devices.

  In order to add the reagent, the microplate reader may be equipped with a corresponding injection device designed to add the reagent to the sample in the wells of the microplate used in the microplate reader. The injector may dispense the reagent, and may cause a chemical reaction in the well currently positioned on the optical axis of the third measuring device and the associated luminescence reaction. The observation (measurement or detection) of a chemical reaction can be performed, for example, almost simultaneously with the addition of the reagent by observing the luminescence reaction, and if necessary, following the same time intervals in the same well. Alternatively or additionally, the observation (measurement or detection) of the chemical reaction may be performed in a different well than the one to which the reagent is currently added, for example by observing the luminescence reaction. In particular, this may be carried out at about the same time as the addition of the reagent in the well, with subsequent time intervals if necessary.

  It is preferable to add reagents to the sample in the wells and simultaneously observe chemical reactions in wells that are separated by a certain distance from this, for example, adjacent wells. In this sense, the reagent is continuously added to the sample in each well of the microplate, and the chemical reaction in wells separated by a certain distance from this, for example, adjacent wells, is simultaneously observed, for example, by a certain distance. It is possible to observe (measure or detect) the luminescence and / or the fluorescence and / or the absorbance of a sample contained in a separate well, for example, the adjacent well. According to this procedure, after another well, "stimulate" one well of the microplate (i.e., add reagents to the sample contained therein), and the chemical reaction caused by the stimulation for a certain time interval. It is possible to observe (measurement or detection) with a delay of The time intervals of this time delay can be determined by, for example, geometric offsets (ie, lateral distances in the XY plane) between individual wells separated by a certain distance, eg, individual adjacent cells, as well as microplates and chemical reactions. The measuring device (more precisely, its optical axis) used to observe is determined by the speed at which it moves relative to one another, in particular in a plane parallel to the microplate.

This object is achieved by the features as disclosed herein for the second aspect. Here, a method is provided for measuring biological cells in a microplate reader with a housing surrounding an internal space. The method comprises the following steps a) to e).
a) Providing a sample compartment separated from the instrument compartment and arranged with a holding device for accommodating at least one microplate. here,
a1) The separation plate is disposed in the housing so as to divide the internal space of the housing into the sample section and the instrument section, or
a2) The inner housing in which the holding device is disposed is disposed in the housing, and the separation plate or the inner housing is the light emitted or influenced by the sample in the wells of the microplate used or inserted in the microplate reader, It has at least one opening designed to pass therethrough from the sample compartment to the instrument compartment.
b) storing at least one microplate provided with wells containing samples in the holding device of the microplate reader;
c) positioning the sample wells of the (these) microplates relative to at least one measuring device of the microplate reader.
d) controlling the temperature of the gas atmosphere in the sample compartment.
e) using at least one measuring device to detect light. This light is
Light emitted by the sample in the wells of the microplate used or inserted in this microplate reader, and / or
-Light affected by the sample through which light passes in the wells of the microplate used or inserted in this microplate reader.

  In this way, the order of the steps, in particular steps b) to e) may be changed. Thus, for example, step d) may be performed before step c) or step b) and / or step e) may be performed before step c) or before step d) .

The method may further comprise one or more of the following steps.
f) Measuring the light influenced by the sample passed by light in the wells of the microplate used in the microplate reader to measure the absorbance of the sample.
g) measuring the light emitted from the light-irradiated sample in the wells of the microplate used in the microplate reader to measure the fluorescence of the sample.
h) measuring the light emitted from the light-irradiated sample in the wells of the microplate used in the microplate reader to measure the luminescence of the sample.

  The predetermined gas atmosphere may be adjusted in the sample compartment, and at least one of the above steps f), g), h) may be performed in the adjusted gas atmosphere of step d) above.

  In the method, the gas flowing into the sample compartment and / or the gas present therein may be mixed and / or circulated using a transfer device arranged in the sample compartment.

In the method, the composition of the gas atmosphere in the sample compartment may be controlled using a control unit. Here, it may flow into the sample compartment through the gas inlet, which is actuated by the control unit. In the method, the proportion of at least one particular gas of the various gases in the gas atmosphere in the sample compartment may be controlled, wherein the particular gas comprises nitrogen (N ₂ ), carbon dioxide (CO ₂ ), It is selected from the group comprising oxygen (O ₂ ), carbon monoxide (CO), hydrogen sulfide (H ₂ S), sulfur dioxide (SO ₂ ). In the method, the humidity and / or temperature of the gas atmosphere in the sample compartment may be controlled using a control unit. In the method, air may be ventilated using a ventilator in the sample compartment.

  In a method of measuring biological cells and / or in the use of a microplate reader (see below), an injection device may be used to add reagents to at least one sample in at least one well of the microplate . A chemical reaction can thus occur, which can be detected using at least one measuring device. In particular, here, together with the chemical reaction, a luminescence reaction of the sample, a change of the luminescence properties, and / or a change of fluorescence and / or a change of absorbance occur. For example, the reagent may be added into the well currently located at the optical axis of the third measuring device, which causes a chemical reaction.

  The observation (measurement or detection) of a chemical reaction can be carried out in the same well, approximately simultaneously with the addition of the reagent, for example, with a subsequent time interval if necessary, by observing the resulting luminescence reaction. Alternatively or additionally, the observation (measurement or detection) of a chemical reaction may be performed in a different well than the one to which the reagent is currently added, for example by observing the resulting luminescence reaction. In particular, this may be performed approximately simultaneously with the addition of the reagents to the wells, with subsequent time intervals if necessary.

  As already pointed out, it is preferable to add reagents to the sample in the wells and simultaneously observe the chemical reaction in the wells separated by a certain distance from this, for example, the adjacent wells. Chemical reactions in a sample (eg, cell culture) produced by the addition of reagents or cell culture observe the rate of growth or change of the sample or cell culture placed in the wells of the microplate (measurement or detection) By doing this, you can observe and identify. In the method, the illumination device may be used to pass light to the sample in the well of the microplate used (inserted) in the microplate reader and / or to illuminate the sample in the well You may

  The sample in the wells of a microplate used in a microplate reader may be illuminated using a second illumination device. The second lighting device may be arranged in the equipment compartment. It may preferably be selected from the group comprising lasers, flash lamps, LEDs (light emitting diodes), laser diodes. In the method, the absorbance of the sample and / or the fluorescence of the sample and / or the luminescence of the sample can each be measured using a measuring device arranged in the sample compartment or the instrument compartment. In particular, the absorbance of the sample can be measured using a first measuring device placed in the sample compartment and / or the fluorescence of the sample can be measured using a second measuring device placed in the instrument compartment. Also, the luminescence of the sample can be measured using a third measuring device arranged in the sample compartment or the instrument compartment.

In a microplate reader in which the method can be carried out, the control unit is an O ₂ sensor for controlling the oxygen content and carbon dioxide content of the gas atmosphere surrounding the sample receiving well of the microplate used (inserted) in this microplate reader And / or a CO ₂ sensor may be provided. Here, the oxygen concentration and / or the carbon dioxide concentration of this gas atmosphere is carbon dioxide when measuring microaerobic, optionally anaerobic and obligately anaerobic microorganisms, fungi, and eukaryotic cells. By selectively accepting nitrogen, it can be maintained at a predetermined value.

In the method described above, the following items d1) to d3) are feasible.
d1) When controlling the composition of the gas atmosphere in the sample compartment, the concentration of the gas already present in the gas atmosphere, eg oxygen (O ₂ ) or carbon dioxide (CO ₂ ), is chemically inert gas, eg , Nitrogen or an inert gas can be reduced by receiving into the sample compartment (this will, in particular, "dilute" or partially squeeze out the gas atmosphere containing the gas already present), and Measuring the concentration of the gas already present by means of a sensor that is responsive to the gas already present, eg an O ₂ sensor and / or a CO ₂ sensor, or
d2) When controlling the composition of the gas atmosphere in the sample compartment, the concentration of the gas already present in the gas atmosphere, eg oxygen (O ₂ ) or carbon dioxide (CO ₂ ) Measuring the concentration of this gas by means of a sensor that can be raised by receiving it (in which, in particular, the concentration of this gas selectively increases), and using a sensor that responds to this gas, Or
d3) When controlling the composition of the gas atmosphere in the sample compartment, the concentration of gases not present or already present in the gas atmosphere, eg hydrogen sulfide (H ₂ S) or carbon monoxide (CO) The concentration of this gas can be increased by receiving it into the sample compartment (thereby, in particular, selectively increasing the concentration of this gas), and using a sensor that responds to this gas. To measure.

  This object mentioned above is achieved, according to the third aspect, by the use according to the invention of the above-mentioned microplate reader according to the invention, or by the above method according to the invention. These uses relate to the measurement of living cells in a microplate reader, living cells including microaerobic, optionally anaerobic and obligately anaerobic microorganisms, fungi, eukaryotic cells Selected from the group.

Additional inventive features are provided by the respective dependent claims. The use of such microplate reader, particularly preferred in measurement microaerophilic or facultative (facultative) anaerobic microorganisms in a given O ₂ concentration. Also preferred is a method of measuring living cells with a microplate reader, wherein the living cells are selected from the group comprising microaerophilic, facultative anaerobic microorganisms, and fungi and eukaryotic cells. Multi-well plates or microtiter plates according to the ANSI / SBS 2004 standard are designated as microplates, for example, with 6, 12, 24, 48, 96, 384 or 1536 wells.

The microplate reader according to the present invention has the following advantages.
The microplate provided with the sample to be measured does not have to be permanently transferred back and forth between the incubator and the measuring device. Thus, such transfers can be omitted if the cells or cell cultures are exposed to the atmosphere and are affected by such atmosphere, resulting in distorted measurement results.
Measurement of luminescence and / or fluorescence and / or absorbance can be performed in a completely automatic way, so that after charging of the microplate reader according to the invention, an operator needs to be present There is no
• In contrast to conventional (eg CO ₂ ) incubators without integrated fluorescence measurements, long-term measurements can be carried out using the microplate reader according to the invention and data can not be detected It can prevent the occurrence of so-called "night windows".
· O ₂ and / or CO ₂ concentration control in an atmosphere or ambient environment above the microplate wells, fine anaerobic and facultative anaerobic The polarization of a given O ₂ and / or CO ₂ concentration Allows measurement of anaerobic microorganisms, fungi, and eukaryotic cells.

  The microplate reader according to the present invention and its use according to the present invention will be described with reference to the schematic drawings showing exemplary and preferred embodiments without limiting the scope of the present invention.

Fig. 1 shows a rather schematic vertical section through a microplate reader according to the invention according to a first preferred embodiment, using a separating plate, comprising an internal space of the housing divided into an instrument compartment and a sample compartment. Fig. 5 shows a rather schematic vertical section through a microplate reader according to the invention according to a second preferred embodiment, using a separating plate, comprising an internal space of the housing divided into an instrument compartment and a sample compartment. Fig. 6 shows a rather schematic vertical sectional view through a microplate reader according to the invention according to a third preferred embodiment, using a separating plate, comprising an internal space of the housing divided into an instrument compartment and a sample compartment. O ₂ sensor in the interior wall surface of the micro plate reader according to the present invention, CO ₂ sensor, an exemplary arrangement of the fan and the gas inlet shown. FIG. 6 shows growth curves of eukaryotic tumor cells in serum-containing medium (MWS) obtained with or without CO ₂ control using the microplate reader according to the present invention. Using a microplate reader according to the present invention, obtained with or without the CO ₂ control, it shows the growth curves of eukaryotic tumor cells in serum free medium (MWOS).

  FIG. 1 shows a rather schematic vertical sectional view through a microplate reader 1 according to the invention according to a first preferred embodiment, wherein an internal space 16 comprises an instrument compartment 18 and a sample compartment 19 using a separating plate 15. And a housing 17 divided into two. This microplate reader 1 comprises at least one measuring device (2 ', 2 ", 2"') for light detection.

  FIG. 1 shows a first measuring device (2 ′) according to a first modification of the microplate reader 1 according to the present invention, using this in the well 3 of the microplate 4 used in this microplate reader 1 The light affected by the sample through which the light has passed can be measured (and thus the absorbance of the sample). This first measuring device 2 'is preferably arranged in the sample section 19 of the microplate reader 1 and has a first illumination device 11' for passing light through the sample in the well 3 of the microplate 4 , Preferably in the instrument compartment 18 of the microplate reader 1. As a result, the first measuring device 2 ′ for detecting light affected by the sample in the well 3 of the microplate 4 used in the microplate reader 1 is the microplate inserted in the microplate reader 1. It is arranged in the direction of the first optical axis 13 'on the second side 14 (i.e. lower side) of the four. As a result, the first illumination device 11 ′ for passing light to the sample is disposed on the first side 12 (ie, the upper side) of the microplate 4 inserted in the microplate reader 1 of the first optical axis 13 ′. Arranged in the direction.

  The assignment of the first illumination device 11 ′ to the first optical axis 13 ′ takes place here by means of the first fiber optic line 33 ′. The first lighting device 11 ′ comprises a first fiber slide 34 ′, a first wavelength selective monochromator 35 ′ and a flash lamp 36. As a result, the light of the flash lamp 36 is guided for transmissive illumination of the sample via the first optical system 23 'to the sample in the direction of the first optical axis 13' using the first fiber optic line 33 '. Ru. The first optical system 23 'can be height adjustable in the direction of the Z axis of the Cartesian coordinate system (see double arrow Z). In this case, the holding device 5 holds the at least one microplate 4 and the holding device 5 for positioning the wells 3 of these microplates 4 with respect to the first measuring device 2 ′ and the first optical axis 13 ′ It is arranged between the device 11 ′ and the first measuring device 2 ′, preferably in the sample compartment 19.

  A second measuring device 2 ′ ′ according to a second variant of the microplate reader 1 according to the invention is shown in FIG. 1, using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light emitted by the sample can be measured with respect to the second optical axis 13 '' (and thus the fluorescence of the sample). The second measuring device 2 ′ ′ includes a photomultiplier tube (PMT) 24, a second wavelength selective monochromator 35 ′ ′, and a second fiber slide 34 ′ ′. For this fluorescence measurement, a microplate reader 1 is provided. The first illumination device 11 ′ is used to illuminate (excite) the sample in the well 3 of the microplate 4.

The assignment of the first illumination device 11 'to the second optical axis 13 "is done in this case using the second fiber optic line 33" and the second optical system 23 ". The second optical system 23" It can be arranged to be adjustable in height in the direction of the Z axis of the Cartesian coordinate system (see double arrow Z). As a result, the light of the flash lamp 36 is guided to illuminate the sample in the direction of the second optical axis 13 "using the second fiber optic line 33". Two different arrangements can be used to detect the fluorescence emitted by the sample.
In so-called “top reading”, the second optical system 23 ′ ′ is used above the microplate 4 and is connected to the second fiber slide 34 ′ ′ using the second fiber optic line 33 ′ ′, As a result, a photomultiplier tube (PMT) 24 can be used to detect the fluorescence of all single samples in well 3 of microplate 4.
In so-called “bottom reading”, the third optical system 23 ′ ′ ′ is below the microplate 4 and the third fiber optic line 33 ′ ′ ′ with the first and second fiber slides 34 ′, 34 The illumination device 11 ′ and the same measurement device 2 ′ ′ can be used to excite and detect the fluorescence of all single samples in the wells 3 of the microplate 4.

  In this case, the holding device 5 is preferably provided with a first or second measuring device 2 in the sample compartment 19 in order to receive at least one microplate 4 and position the wells 3 containing the samples of these microplates 4. For ', 2', it is preferably located below the first lighting device 11 'housed in the equipment compartment 18.

  A third measuring device 2 ′ ′ ′ according to a third modification of the microplate reader 1 according to the present invention is shown in FIG. 1 and using this in the well 3 of the microplate 4 used in this microplate reader 1 The light emitted by the sample can be measured at (e.g. luminescence of the sample). A third measuring device 2 "'is used for this luminescence measurement, which is preferably a microplate reader 1 The generally known injection device 10 reaching the sample compartment 19 of the microplate reader 1 is preferably arranged in the instrument compartment 18 of the third embodiment with respect to the third optical axis 13 ′ ′ ′. Cause a chemical reaction involving a luminescence reaction or change in luminescence characteristics and / or a change in fluorescence and / or a change in absorbance in a sample The injection device 10 is preferably arranged at this point in time with the well 3 on the third optical axis 13 "'and thus precisely above the third measuring device 2"'. In this case, a reagent that causes a chemical reaction, for example, a luminescence reaction, is configured and arranged so that it can be added to the well 3 of the microplate 4.

  A preferred embodiment of the microplate reader 1 is designed such that reagents are added to the sample in well 3 and chemical reactions in wells that are some distance apart from this, eg adjacent wells, are simultaneously observed. In this sense, the reagent is continuously added to the sample in each well 3 of the microplate 4 and the chemical reaction in the wells separated by a certain distance from this, for example, adjacent wells, is simultaneously observed, for example, It is possible to observe (measure or detect) luminescence and / or fluorescence and / or absorbance of a sample accommodated in wells separated by a distance, for example, adjacent wells. According to this procedure, after another well, "stimulate" one well of the microplate (i.e., add reagents to the sample contained therein), and the chemical reaction caused by the stimulation for a certain time interval. It is possible to observe (measurement or detection) with a delay of The time intervals of this time delay are: geometry offsets (i.e., lateral distances in the XY plane) between wells (e.g., next to each other) separated by individually related wells, as well as microplate 4 and chemical reaction Determined by the speed at which the measuring devices 2 ′, 2 ′ ′, 2 ′ ′ ′ (or their optical axes 13 ′, 13 ′ ′, 13 ′ ′ ′) used for observation move relative to one another, in particular relative to the XY plane Be done.

  A corresponding injection device 10 is envisaged in the embodiment of the microplate reader shown in FIGS. 2 and 3, the one described above for the injection device 10 and its function and use apply to the embodiment shown here.

  The third measuring device 2 ′ ′ ′ preferably comprises a fourth optical system 23 ′ ′ ′ ′, which can be arranged movably in the direction of the Z axis of the Cartesian coordinate system (see double arrow Z). The second, third and fourth optical systems 23 ′, 23 ′ ′, 23 ′ ′, 23 ′ ′ ′ ′ can be partially lowered into the sample compartment 19 through the individual openings 20 of the separating plate 15 in a particularly preferred manner It is arranged like.

The separation control unit 6 'is disposed on the housing 17 of the microplate reader 1 according to the present invention, and the arrangement of the O ₂ sensor 8, the CO ₂ sensor 9, the fan 22 and the gas inlet 21 is the microplate according to the present invention It is provided inside the rear wall 30 of the housing 17 of the reader 1 (see FIG. 4). The control unit 6 ′ of the microplate reader 1 preferably measures the oxygen content of the gas atmosphere 7 around the sample compartment 19, ie the well 3 containing the samples of the microplate inserted in said microplate reader 1 An O ₂ sensor 8 for controlling is provided. In a particularly preferred manner, the control unit 6 of the microplate reader 1 substitutes or adds to the O ₂ sensor 8 a gas atmosphere 7 surrounding a well 3 containing a sample of the microplate 4 inserted in said microplate reader 1. A CO ₂ sensor 9 is provided to measure and control the carbon dioxide content. Thus, the control unit 6 is arranged partially and externally inside and on the housing 17 of the microplate reader 1 and can be connected or connectable to the microplate reader 1 via the electrical contacts 27 and the gas line 38 . The separation control unit 6 'is directly connected with the necessary pressure cylinder for the gas used. Individual valves and throttles for the required gas connections are installed in the separation control unit 6 '(not shown). Alternatively, it is also possible to use a mouse and a throttle on the gas pressure cylinder, which are preferably arranged as electrically controlled solenoid valves (type of normally closed). FIG. 1 does not show, among other things, control elements, display elements, power supplies for the separation control unit 6 ′ and the microplate reader 1.

  FIG. 2 shows a rather schematic vertical sectional view through the microplate reader 1 according to the invention according to a second preferred embodiment, wherein the internal space 16 comprises an instrument compartment 18 and a sample compartment 19 using a separating plate 15. The housing 17 is divided into two. The microplate reader 1 comprises at least one measuring device 2 ', 2 ", 2"' for light detection.

  FIG. 2 shows a first measuring device 2 ′ according to a first variant of the microplate reader 1 according to the invention, using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light affected by the sample through which the light has passed can be measured (and hence the absorbance of the sample). This first measuring device 2 'is preferably arranged in the sample section 19 of the microplate reader 1 and has a first illumination device 11' for passing light through the sample in the well 3 of the microplate 4 , Preferably in the instrument compartment 18 of the microplate reader 1. As a result, the first measuring device 2 ′ for detecting light affected by the sample in the well 3 of the microplate 4 used in the microplate reader 1 is the microplate inserted in the microplate reader 1. It is arranged in the direction of the first optical axis 13 'on the second side 14 (i.e. lower side) of the four. As a result, the first illumination device 11 ′ for passing light to the sample is disposed on the first side 12 (ie, the upper side) of the microplate 4 inserted in the microplate reader 1 of the first optical axis 13 ′. Arranged in the direction.

  The assignment of the first lighting device 11 'to the first optical axis 13' is done in this case using a partially transparent (e.g. 50%) mirror 26 or a dichroic mirror. The first lighting device 11 ′ includes a first wavelength selection filter 37 ′ and a flash lamp 36. As a result, the light of the flash lamp 36 is guided for transmitted illumination of the sample via the first optical system 23 'to the sample in the direction of the first optical axis 13' using the partially transparent mirror 26. The first optical system 23 'can be height adjustable in the direction of the Z axis of the Cartesian coordinate system (see double arrow Z). In this case, the holding device 5 holds the at least one microplate 4 and the holding device 5 for positioning the wells 3 of these microplates 4 with respect to the first measuring device 2 ′ and the first optical axis 13 ′ It is arranged between the device 11 ′ and the first measuring device 2 ′, preferably in the sample compartment 19.

  A second measuring device 2 ′ ′ according to a second variant of the microplate reader 1 according to the invention is shown in FIG. 2 and using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light emitted by the sample can be measured with respect to the first optical axis 13 'or the second optical axis 13' '(and thus the fluorescence of the sample). The second measurement device 2 ′ ′ includes a photomultiplier tube (PMT) 24 and a second wavelength selection filter 37 ′ ′. For this fluorescence measurement, the first illumination device 11 ′ of the microplate reader 1 is again used to illuminate (excite) the sample in the well 3 of the microplate 4.

  The assignment of the first illumination device 11 'to the second optical axis 13' 'is done in this case using a partially transparent (e.g. 50%) mirror 26 or a dichroic mirror and the first optical system 23'.

Two different arrangements can be used to detect the fluorescence emitted by the sample.
In so-called “bottom reading”, the second optical system 23 ′ ′ is below the microplate 4 and is connected to the partially transparent mirror 26 and the second filter 37 ′ ′ using the first fiber optic line 33 ′ As a result, photomultiplier tubes (PMTs) 24 can be used to detect the fluorescence of all single samples in well 3 of microplate 4.
In so-called "top reading", the first optical system 23 'is used above the microplate 4 and is connected to the second filter 37 "using the partially transparent mirror 26, so that the same A photomultiplier tube (PMT) 24 can be used to detect the fluorescence of all single samples in well 3 of microplate 4.

  In this case, the holding device 5 is preferably provided with a first or second measuring device 2 in the sample compartment 19 in order to receive at least one microplate 4 and position the wells 3 containing the samples of these microplates 4. For ', 2', it is preferably located below the first lighting device 11 'housed in the equipment compartment 18.

  A third measuring device 2 ′ ′ ′ according to a third variant of the microplate reader 1 according to the invention is shown in FIG. 2 and using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light emitted by the sample can be measured at (e.g. luminescence of the sample). A third measuring device 2 "'is used for this luminescence measurement, which is preferably a microplate reader 1 The generally known injection device 10 reaching the sample compartment 19 of the microplate reader 1 is preferably arranged in the instrument compartment 18 of the third embodiment with respect to the third optical axis 13 ′ ′ ′. Cause a chemical reaction involving a luminescence reaction or change in luminescence characteristics and / or a change in fluorescence and / or a change in absorbance in a sample The injection device 10 is preferably arranged at this point in time with the well 3 on the third optical axis 13 "'and thus precisely above the third measuring device 2"'. In this case, a reagent that causes a chemical reaction, for example, a luminescence reaction, is configured and arranged so that it can be added to the well 3 of the microplate 4.

  The third measuring device 2 ′ ′ preferably comprises a third optical system 23 ′ ′, which can be arranged movably in the direction of the Z axis of the Cartesian coordinate system (see double arrow Z). The first and second optical systems 23 ′, 23 ′ ′ are arranged in a particularly preferred manner in such a way that they can be partially lowered into the sample compartment 19 via the individual openings 20 of the separating plate 15.

  The same control unit 6 is preferably used in all the aforementioned variants of the microplate reader 1 according to the invention.

  FIG. 3 shows a rather schematic vertical sectional view through a microplate reader according to the invention according to a third preferred embodiment, wherein an internal space 16 is provided by means of an internal housing 39 into an instrument compartment 18 and a sample compartment 19. The housing 17 is divided. The microplate reader 1 comprises at least one measuring device 2 ', 2 ", 2"' for light detection. The inner housing 39 is provided with an opening 20, which allows the light emitted in the well 3 of the microplate 4 used in the microplate reader 1 or the light influenced by the sample to pass through it from the sample compartment 19 to the instrument It is designed to reach the compartment 18.

  FIG. 3 shows a first measuring device 2 ′ according to a first variant of the microplate reader 1 according to the invention, using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light affected by the sample through which the light has passed can be measured (and hence the absorbance of the sample). This first measuring device 2 ′ is preferably arranged in the instrument section 18 of the microplate reader 1 and has a first illumination device 11 ′ for passing light through the sample in the wells 3 of the microplate 4 Preferably, it is arranged in the sample compartment 19 of the microplate reader 1. As a result, the first measuring device 2 ′ for detecting light affected by the sample in the well 3 of the microplate 4 used in the microplate reader 1 is the microplate inserted in the microplate reader 1. It is arranged in the direction of the first optical axis 13 'on the first side 12 (i.e. the upper side) of the four. As a result, the first illumination device 11 'for transmitting light to the sample is disposed on the second side 14 (ie, the lower side) of the microplate 4 inserted in the microplate reader 1 and the first optical axis 13'. Placed in the direction of

  The first measuring device 2 ′ preferably comprises a mirror 25 for deflecting the light coming from the sample in the direction of the photomultiplier or PMT 24. Alternatively, light coming from the sample can be provided using PMT's 24 fiber optics (not shown). Similarly, the first lighting device 11 'can be arranged as a lamp and can be arranged on the first optical axis 13'. Alternatively, the light for passing light through the sample can be guided in the direction of the first optical axis 13 'using mirrors or fiber optics (both not shown). In this case, a holding device 5 is preferably provided for accommodating the at least one microplate 4 and for positioning the wells 3 containing the samples of these microplates 4, preferably the first illumination device relative to the first measuring device 2 '. It is arranged between 11 ′ and the first measuring device 2 ′, preferably in the sample compartment 19.

  A second measuring device 2 ′ ′ according to a second variant of the microplate reader 1 according to the invention is shown in FIG. 3 and using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light emitted by the sample can be measured (and thus the fluorescence of the sample) For this fluorescence measurement, the first measuring device 2 'of the microplate reader 1 is again used, in this case the second measuring device 2 " Is the same as A second illumination device 11 '' (preferably a laser, a flash lamp or a laser diode) is used as a light source for the excitation light needed to illuminate the sample in the wells 3 of the microplate 4. 11 ′ ′ are preferably arranged in the housing space 18 of the microplate reader 1. As a result, both the first measuring device 2 ′ and the second illumination device 11 ′ ′ for the detection of the light emitted by the sample in the wells 3 of the microplate 4 inserted in this microplate reader 1 are Located on the same side 12 (and thus the upper side) of the microplate 4 inserted in the reader 1. The second illumination device 11 ′ ′ preferably directs the excitation light in the direction of the first optical axis 13 ′, ie towards the sample A partially transparent (eg, 50%) mirror 26 or a dichroic mirror for deflecting is provided. In this case, the holding device 5 is preferably arranged relative to the first measuring device 2 ′ in the sample compartment 19 in order to receive at least one microplate 4 and position the wells 3 containing the samples of these microplates 4. It is preferably located below the second lighting device 11 ′ ′ and the first measuring device 2 ′ housed in the equipment compartment 18.

  A third measuring device 2 ′ ′ ′ according to a third variant of the microplate reader 1 according to the invention is shown in FIG. 3 and using this in the wells 3 of the microplate 4 used in this microplate reader 1 The light emitted by the sample can be measured at (e.g. luminescence of the sample). A third measuring device 2 "'is used for this luminescence measurement, which is preferably a microplate reader 1 The generally known injection device 10, which is preferably arranged in the sample compartment 19 of the microplate reader 1 in the sample compartment 19 of the Cause a chemical reaction that involves the luminescence reaction or changes in luminescence characteristics, changes in fluorescence, and / or changes in absorbance. The injection device 10 is, for example, a chemical reaction when the well 3 is then placed on the second optical axis 13 ′ ′ and thereby correctly placed above the third measuring device 2 ′ ′ For example, a reagent that causes a luminescence reaction is configured and arranged so that it can be added to the well 3 of the microplate 4.

  In this case, the holding device 5 is preferably arranged relative to the second measuring device 2 ′ ′ in the sample compartment 19 in order to house the at least one microplate 4 and position the wells 3 containing the samples of these microplates 4. It is disposed below the injection device 10 and above the third measuring device 2 ′ ′ ′. The first measuring device 2 'preferably comprises a first optical system 23', which can be arranged movably in the direction of the Z axis of the Cartesian coordinate system. In a particularly preferred manner, the first optical system 23 ′ can be partially lowered into the sample compartment 19 via the individual openings 20 in the inner housing 39 according to the first embodiment as shown in FIG. Be placed.

  The microplate 4 and the holding device 5 have first, second and / or third optical axes 23 ', 23 ", 23 of the first, second and / or third measuring devices 2', 2", 2 "'. For '', it is movable in the direction of the Z axis of the Cartesian coordinate system (see double arrow Z in FIG. 2, FIG. 3). Microplate 4 or holding device 5 (see double arrows X, Y) and / or first, second and / or third measuring devices 2 ', 2 ", 2"' or their first, second and The third optical axis 23 ′, 23 ′ ′, 23 ′ ′ ′ may be designed to be movable in the direction of the X axis and / or the Y axis of the Cartesian coordinate system. This is applicable to all the illustrated embodiments and variations and possible modifications of the microplate reader 1 according to the invention.

  In all the embodiments and modifications of the microplate reader 1 according to the present invention, the microplate reader 1 has the composition of the gas atmosphere 7 surrounding the well 3 containing the sample of the microplate 4 inserted in the microplate reader 1. And a control unit 6 for controlling the Furthermore, the holding device 5 is preferably arranged to be movable in the direction of the X and Y axes of the Cartesian coordinate system (see double arrows X, Y in FIGS. 1 to 3).

FIG. 4 shows an exemplary arrangement of the O ₂ sensor 8, the CO ₂ sensor 9, the fan 22 and the gas inlet 21 inside the rear wall 30 of the housing 17 of the microplate reader 1 according to the invention. The control unit 6 of the microplate reader 1 is preferably an O ₂ sensor for measuring and controlling the oxygen content of the gas atmosphere 7 surrounding the well 3 containing the sample of the microplate 4 inserted in this microplate reader 1 It has eight. Microplate reader 1 of the control unit 6, in a particularly preferred method, the O ₂ as an alternative or additional sensor 8, a gas atmosphere 7 surrounding the wells 3 for accommodating a sample of the microplate 4 inserted in this microplate reader 1 And a CO ₂ sensor 9 for measuring and controlling the carbon dioxide content of the The control unit 6 is partially locatable outside the housing 17 of the microplate reader 1 and is connected or connected to the microplate reader 1 (not shown) via electrical contacts 27 and a gas conduit (not shown) It is possible.

  However, it is also possible to provide that the control unit 6 is completely integrated in the microplate reader 1 and housed in the common housing 17 (see FIGS. 2 and 3). In this case, the microplate reader 1 will be connected directly with the necessary pressure cylinder for the gas used (see FIGS. 2 and 3). Preferably, the individual valves and throttles are installed in the housing 17 of the microplate reader 1 (not shown). Alternatively, it is also possible to use valves and throttles in the gas pressure cylinder, which are preferably arranged as electrically controlled solenoid valves (type of normally closed) (not shown).

The control unit preferably comprises a computer 28 with individual software. In this case, the computer 28 can be connected to the central computer 29 of the microplate reader 1 (see FIG. 1) (for example housed in a separate housing 17 ') or integrated in the central computer 29 of the microplate reader 1 it can. Preferably, nitrogen (N ₂ ) and / or carbon dioxide (CO ₂ ) used to replace the ambient air is provided in the pressure cylinder and widely known to set the required supply pressure for these gases. Control valves and throttles are used in these pressure cylinders. Such process gases may alternatively be obtained from other sources (eg, from in-house conduits).

  In addition to nitrogen and carbon dioxide, other gases can be used to generate a predetermined proportion of the atmosphere that normally prevails in the sample compartment (in combination with the individual gas detectors in the sample compartment 19). By means of microplates 4 inserted in the area of wells 3 in the microplate reader 1 in order to produce a specific composition of the gas atmosphere by following the potentially applicable safety measures. Other noble gases or inert gases (eg argon) or reactive or toxic gases (eg oxygen, carbon monoxide, hydrogen sulfide, sulfur dioxide) in the sample compartment 19 of the microplate reader 1 It is also possible to introduce Preferably, the control unit 6 is equipped with sufficient gas conduits and gas valves so that a more complex gas composition with multiple gas components can be realized.

For the appropriate CO ₂ sensor, we refer to the currently used CO ₂ sensor (SenseAir CO ₂ Engine ICB, Part No .: 033-9-0001 of SenseAir AB in SE-820 60 Delsbo, Sweden). Respect Suitable O ₂ sensor, refer to the O ₂ sensor currently in use (Pewatron FCX-MEP2-F- CH oxygen module of Pewatron AG in CH-8052 Zurich, Switzerland).

The control unit 6 can control the oxygen content of the gas atmosphere 7 surrounding the well 3 containing the sample of the microplate 4 inserted into the microplate reader 1 in combination with the O ₂ sensor, as a result, The microplate reader 1 can be used to measure microaerophilic or facultative anaerobic microorganisms under a predetermined concentration of O ₂ . Measurement of anaerobic microorganisms or eukaryotic cells is possible under a predetermined concentration of O ₂ .

The control unit 6 can control, in combination with a CO ₂ sensor, the carbon dioxide content of the gas atmosphere 7 surrounding the well 3 containing the sample of the microplate 4 inserted in this microplate reader 1, as a result The microplate reader 1 can be used in the measurement of living cell culture under predetermined CO ₂ concentration.

  In the context of the present invention, cell cultures, biological cell stores isolated from such cell cultures, separately obtained cells or individual cells are designated as cells comprising microorganisms, fungi, animal and plant eukaryotic cells. It will be

  As shown in FIGS. 1-4, any combination of elements of microplate reader 1 and control unit 6 is within the scope of the present invention.

The 5% CO ₂ modulation of eukaryotic tumor cells was tested using the microplate reader 1 of the first embodiment according to the present invention (see FIG. 1). GFP transfected A431 cells were used for fluorescence measurements. The abbreviation GFP refers to the widely known "green fluorescent protein" which has an excitation maximum at wavelengths of 396 nm and 475 nm and an emission maximum at wavelength of 508 nm. These cells were suspended in 10% serum-supplemented (MWS) or serum-free (MWOS) media to record individual growth curves for 72 hours. Serum-containing medium (MWS) had the following composition. Dulbecco's modified Eagle's medium (DMEM with phenol red), supplemented with 4.5 g / l glucose, 10 mM HEPES buffer, 2 mM L-glutamate, 1 mM Na-pyruvate, 100 U / ml penicillin, 0.1 mg / ml ml Streptomycin, 5% (v / v) fetal bovine serum (FBS). All media components were obtained from PAA laboratories (Linz, Austria).

  Serum free medium (MWOS) contained the same components except for fetal bovine serum (FBS).

  Greiner standard cell culture MTP 96 well Microplate 4 of sterile coated type (Greiner 96 well, black, flat & clear bottom, sterile-culture tissue plates) was used. In 48 wells 3 of these microplates 4, 2500 A431 GFP cells and 10% serum were respectively injected (sample type A, see FIG. 5). The remaining 48 wells 3 of these microplates 4 were each loaded with 2500 A431 GFP without serum (sample type B, see FIG. 6). Two test series were performed using these two sample types A and B.

The first test series is to measure the concentration of CO ₂ in the sample compartment 19 using a CO ₂ sensor (SenseAir, Typ CO ₂ Engine ICB, Part No .: 033-9-0001). The control unit 6 with integrated electronic control system was kept constant. This is because the atmosphere in the sample compartment 19 and the equipment compartment 18 of the microplate reader 1 according to the invention initially has a gas composition corresponding to one of the ambient air (ie approximately corresponding to the standard atmosphere) So achieved. The standard atmosphere has the following composition. Oxygen 20.93%, nitrogen 78.10%, argon 0.93%, carbon dioxide 0.03%, hydrogen, neon, helium, krypton, xenon 0.01%.

The housing 14 of the microplate reader 1 is sealed after positioning of the microplate 4 in the holding device 5 of the microplate reader 1, and the CO ₂ gas is introduced into the gas inlet 21 until the CO ₂ gas concentration reaches 5%. Into the sample compartment 19. This CO ₂ concentration remained unchanged during the measurement period, and CO ₂ gas was introduced into the sample compartment 19 as needed (every time the CO ₂ sensor determined that the carbon dioxide concentration was insufficient). The gas mixture in the sample compartment 19 was constantly circulated using a fan. The temperature was kept constant at 37 ° C. in each case.

The second test series is to permanently expose the sample to ambient air without introducing additional CO ₂ into the sample compartment 19. The temperature was kept constant at 37 ° C. in each case.

  The fluorescence of each sample (each well 3) is excited using the first illumination device 11 'disposed below the microplate 4 (excitation at λ = 485 nm, fluorescence detection at λ = 535 nm), and the second optical The system 23 ′ ′ was used to guide to a photomultiplier tube (PMT) 24 for detection. Individual fluorescence of individual samples was measured in a time dependent manner. Each single detector detected by the photomultiplier tube 24. The individual fluorescence intensities of one well were processed by central computer 29 of microplate reader 1 and shown in the curve diagram (see FIGS. 5 and 6).

FIG. 5 shows growth curves of eukaryotic tumor cells in 10% serum-containing medium (MWS) obtained with or without CO ₂ control using the microplate reader according to the present invention. Reference 31 represents a sample in which the CO ₂ concentration remained unchanged at 5%, and reference 32 represents a sample exposed to normal ambient air.

FIG. 5 shows that for the first 35 hours, the cells were almost as well activated and the cell viability of sample 31 (where the CO ₂ concentration was kept unchanged at 5% (based on the corresponding GFP signal (ie (Measured based on the intensity of fluorescence), but slightly lower than the cell viability of the sample 32 exposed to normal ambient air. After about 35 hours, the growth of cells of sample 32 exposed to normal ambient air was dramatically reduced. It reached a maximum of about 450% after about 60 hours and decreased to less than 400% of the initial value at the end of the 75 hour measurement.

On the other hand, the cell viability of the sample 31 in which the CO ₂ concentration is kept unchanged at 5% gradually increases linearly after the measurement period of 35 hours, and at the end of the 75 hours of measurement, the initial value is about 900. Reached%.

FIG. 6 shows growth curves of eukaryotic tumor cells in 10% serum free medium (MWOS) obtained with or without CO ₂ control using the microplate reader according to the present invention. Reference 31 represents a sample in which the CO ₂ concentration remained unchanged at 5%, and reference 32 represents a sample exposed to normal ambient air.

FIG. 6 shows that for the first 35 hours, the cells were almost as well activated and the cell viability of sample 31 (where the CO ₂ concentration was kept unchanged at 5% (based on the corresponding GFP signal (ie (Measured based on the intensity of fluorescence), but slightly lower than the cell viability of the sample 32 exposed to normal ambient air. After about 35 hours, the growth of cells of sample 32 exposed to normal ambient air reached a maximum of 200% and dropped to an initial value of almost 100% at the end of the 75 hour measurement.

On the other hand, the cell viability of the sample 31 in which the CO ₂ concentration is kept unchanged at 5% increases by almost the same amount after the measurement period of 35 hours, and is about 300% of the initial value at the end of 75 hours of measurement Reached.

The results shown indicate that cells maintained unchanged at a 5% CO ₂ concentration can exhibit substantially higher cell viability than cells maintained in normal ambient air. This greater cell viability plays a role, especially after more than 40 hours of culture time. A further addition of 10% serum doubles cell viability for the first 35 hours and increases three times at 75 hours.

Such uninterrupted, long-term studies are practically possible with the present invention, for example, integrated fluorescence, which needs to take into account the "night windows" of about 14 hours without collectable data. It differs from the conventional CO ₂ incubator without measurement.

  Known from the prior art, comprising a sensor and control unit for controlling the composition of the gas atmosphere above the well containing the sample of the microplate used in this microplate reader or the surrounding environment in the well There is no microplate reader.

  The German published application DE 10 2005 033 927 A1 discloses an illumination device for brightfield transmitted light contrast in an inverted microscope for observing living cells, this illumination device being a closed light shield of an inverted microscope Individual wells of the sample holder, integrated in a flexible compact incubator and arranged as a microtiter plate, illuminated continuously, while at the same time temperature stabilization of the sample volume in combination with minimal heat loss It will be secured for a long time. The illumination device is arranged stationary above the projection lens at the top of the incubator. The inserted microplate, petri dish or the like can be moved relative to the projection lens on a microscope stage located at the bottom of the incubator.

The incubator comprises a first chamber completely surrounded by an outer second chamber, which is thermally isolated from the surrounding environment. Microtiter plates with the sample, temperature, air humidity and CO ₂ content is disposed is controllable in a controlled manner by the control device the inner chamber. This German published application DE 10 2005 033 927 A1 relates exclusively to an inverted microscope, the construction and use of which is quite different from a microplate reader which has no imaging function and which allows a rather short measurement period per well. A microplate reader, in particular an O ₂ sensor, is not mentioned in this publication.

A climate chamber for observing samples in microtiter plates is known from patent EP 1 575 706 B1. A conditioning medium flow in the form of air having a predetermined air humidity and / or temperature can be introduced into the climate chamber. A predetermined introduction of CO ₂ gas and an arrangement of temperature sensors, humidity sensors and / or gas sensors in close proximity to the sample holder carrying the microtiter plate can be provided. A microplate reader, in particular an O ₂ sensor, is not mentioned in this publication.

  As a result, the prior art comprises a control unit 6 for controlling the composition of the gas atmosphere 7 above the well 3 containing the sample of the microplate used in this microplate reader 1 or the surrounding environment in the well The microplate reader 1 according to the present invention is not obvious to a person skilled in the art.

A further preferred application of the microplate reader 1 and the control unit 6 according to the invention is, for example, testing of the effect of O ₂ partial pressure on the growth of microorganisms, eg Rhodospirillum rubrum (literature: Biedermann et al. 1967). , “Archiv fur Mikrobiologie” (Archive for Microbiology 56, 133-147) and a test for microaerobic alginic acid production, preferably an O ₂ concentration of 2.5 to 5% (preferably 2.5%) is set (Reference: Wael Sabra 1999: Microaerophilic alkaline production with Azotobacter vinelandii; Dissertation at the Common Scientific Faculty of the Braunschweig University of Technology).

  The same or corresponding features of the microplate reader 1 shown in the figures are given the same reference numerals if not explicitly referred to in the description. The features of the illustrated embodiments and any combination of technical equivalents of these features are within the scope of the invention disclosed herein.

DESCRIPTION OF SYMBOLS 1 microplate reader 2 '1st measuring apparatus 2''2nd measuring apparatus 2''' 3rd measuring apparatus 3 well 4 microplate 5 holding apparatus 6 control unit 6 'isolation | separation control unit 7 gas atmosphere 8 O ₂ sensor 9 CO ₂ sensor DESCRIPTION OF SYMBOLS 10 injection apparatus 11 '1st illuminating device 11''2nd illuminating device 12 1st side of microplate 4 inserted 13' 1st optical axis 13 '' 2nd optical axis 13 '''3rd optical axis 14 micro inserted 2nd side of plate 4 15 separation plate 16 internal space 17 reader housing 17 'separation housing 18 equipment compartment 19 sample compartment 20 opening 21 gas inlet 22 fan 23' first optical system 23 '' second optical system 23 '''third Optical system 24 PMT photomultiplier tube 25 mirror 26 partial transparent mirror 27 electrical contact 28 Computer 29 microplate cell growth curve of the central computer 30 rear wall 31 CO ₂ control there leader 32 CO ₂ control without cell growth curve 33 'the first fiber optic line 33 "second fiber optic line 33"' third fiber optic Line 34 '1st fiber slide 34''2nd fiber slide 35' 1st monochromator 35 '' 2nd monochromator 36 flash lamp 37 '1st filter 37''2nd filter 38 gas line 39 inner housing

Claims (23)

A microplate reader (1),
a) at least one measuring device (2 ′, 2 ′ ′, 2 ′ ′ ′) selected from the group comprising a1) to a3) below:
a1) designed to measure the absorbance of a sample which is allowed to pass light using the first illumination device (11 ') in the well (3) of the microplate (4) inserted in the microplate reader (1) First measuring device (2 ').
a2) designed to measure the fluorescence of the sample illuminated by the light of the first illumination device (11 ') in the well (3) of the microplate (4) inserted in the microplate reader (1) Second measuring device (2 '').
a3) A third measuring device (2 ′ ′ ′) designed to measure the luminescence of the sample in the wells (3) of the microplate (4) inserted in the microplate reader (1).
b) During measurement with at least one measuring device (2 ′, 2 ′ ′, 2 ′ ′ ′), accommodate and hold at least one microplate (4) containing the sample in the well (3) A holding device (5) configured as
c) Alternative as separation plate (15) or inner housing (39), which divides the interior space (16) of the housing (17) of the microplate reader (1) into an instrument compartment (18) and a sample compartment (19) Division means configured in
The sample compartment (19) is separated from the instrument compartment (18) in a substantially gas tight manner.
The dividing means is fixedly installed in the housing (17),
In the wells (3) of the microplate (4) inserted in the microplate reader (1), light which has been illuminated or influenced by the sample passes through it from the sample compartment (19) to the instrument compartment (18) A dividing means comprising an opening (20) designed to reach;
d) a control unit (6, 6 ') for controlling the temperature of the gas atmosphere (7) in the sample compartment (19), comprising a cooling device for cooling the gas atmosphere in the sample compartment And (6, 6 ') and
Furthermore, at least one first illumination device (11 ′) comprising a flash lamp (36), a first fiber optic line (33 ′) and a first wavelength selective monochromator (35 ′),
The first illumination device (11 ′) is configured to pass or illuminate light of a selected wavelength to the sample in the well (3) of the microplate (4) inserted in the microplate reader (1),
The first illumination device (11 ′) of the microplate reader (1) comprises at least a first optical system (23 ′) mounted on the wall of the opening (20) or the inner housing (39) of the separating plate (15) ,
The holding device (5) is configured to position the sample receiving well (3) of the at least one microplate (4) relative to the at least one measuring device (2 ′, 2 ′ ′, 2 ′ ′ ′) A holding device (5) is movable relative to the dividing means and its opening, at least in the direction of an axis of the Cartesian coordinate system, the holding device (5) being placed in the sample compartment (19) as a whole , Be surrounded by the gas atmosphere in the sample compartment (19) ,
The cooling device is mounted at the bottom of the sample compartment (19) and is in heat exchange communication with the gas atmosphere in the sample compartment (19),
The instrument compartment (18) is arranged above the sample compartment (19), microplate reader (1).   The sample compartment (19) is separated from the apparatus compartment (18) substantially light-tight by means of the separating plate (15) or the inner housing (39), the protection device extending around the opening (20) being light-shielding The microplate reader (1) according to claim 1, characterized in that it is provided for airtight separation.   The microplate reader (1) according to claim 1, characterized in that the control unit (6, 6 ') comprises a heating device for heating the sample compartment. A microplate reader (1) according to claim 3 , characterized in that the heating device is mounted on the dividing means and is in heat exchange communication with the gas atmosphere in the sample compartment. The control unit (6, 6 '), the micro plate reader according to claim 1 Symbol mounting, characterized in that it comprises a temperature sensor for measuring the temperature of the gas atmosphere (1).   The microplate reader (1) according to claim 1, characterized in that the control unit (6, 6 ') is designed to further control the humidity of the gas atmosphere in the sample compartment. A microplate reader (1) according to claim 6 , characterized in that the control unit (6, 6 ') comprises a humidity sensor for measuring the humidity of the gas atmosphere in the sample compartment.   The control unit (6, 6 ') is configured to control the composition of the gas atmosphere (7) in the sample compartment (19), the control unit (6, 6') being installed in the sample compartment (19) The microplate reader (1) according to claim 1, characterized in that it comprises a gas sensor for measuring at least one gas of the gas atmosphere in the sample compartment (19). The microplate reader (1) according to claim 8 , characterized in that the control unit (6, 6 ') comprises a gas sensor for measuring different gases in the sample compartment (19).   A transfer device for mixing and / or circulating the gas present in the sample compartment (19) and / or the gas flowing into the sample compartment (19) is characterized in that it is arranged in the sample compartment (19) The microplate reader (1) according to claim 1. The transfer device is characterized in that it comprises at least one of a blower (22), one or more baffles, a stirrer comprising one or more paddles, or a structured jet nozzle system. The microplate reader (1) according to claim 10 .   The microplate reader (1) according to claim 1, characterized in that it comprises first, second and third measuring devices (2 ', 2 ", 2"').   The first, second or third measuring device (2 ′, 2 ′ ′, 2 ′ ′ ′) comprises a first, second or third optical system (23 ′, 23 ′ ′, 23 ′ ′ ′) and their optics At least one of the systems (23 ', 23 ", 23"') and / or the holding device (5) and / or the inserted microplate (4) can be moved relative to one another in the direction of the Z axis of the Cartesian coordinate system The microplate reader (1) according to claim 1, characterized in that it is designed to   The microplate reader (1) according to claim 1, characterized in that the first optical system (23 ') in the aperture (20) of the dividing means is adjustable in height in the direction of the Z axis of the Cartesian coordinate system. .   The second and / or third measuring devices (2 ′ ′, 2 ′ ′ ′) respectively include second and / or third optical systems (23 ′, 23 ′ ′, 23 ′ ′ ′), and the first, second and third At least one of the three optical systems (23 ′, 23 ′ ′, 23 ′ ′ ′) and / or the holding device (5) are characterized in that they are designed to be movable relative to one another in the direction of the Z axis of the Cartesian coordinate system The microplate reader (1) according to claim 1. A method of measuring living cells in a microplate reader (1) comprising a housing (17) surrounding an internal space (16), comprising:
a) Providing a microplate reader (1) according to claim 1 provided with a cooling device,
b) storing in the holding device (5) of the microplate reader (1) at least one microplate (4) provided with wells (3) for containing samples;
c) Using a holding device (5), the sample storage well (3) of this microplate (4) is placed on at least one measuring device (2 ', 2 ", 2"') of this microplate reader (1) Positioning with respect to
d) controlling the temperature of the gas atmosphere (7) in the sample compartment (19);
e) using at least one measuring device (2 ′, 2 ′ ′, 2 ′ ′ ′) for detecting light, this light being
The light emitted by the sample in the wells (3) of the microplate (4) inserted in this microplate reader (1), and / or
-In the wells (3) of the microplate (4) inserted into this microplate reader (1), the step of being light affected by the sample passed by the light;
The microplate (4) is held by the holding device (5) during the measurement with the at least one measuring device (2 ′, 2 ′ ′, 2 ′ ′ ′),
A method in which the gas atmosphere in the sample compartment is cooled by a cooling device. 17. The method of claim 16 , further comprising at least one or more of the following steps f), g), h).
f) Measuring the light influenced by the sample passed by light in the wells (3) of the microplate (4) inserted in the microplate reader (1) to measure the absorbance of the sample.
g) measuring the light emitted from the light-irradiated sample in the wells (3) of the microplate (4) inserted in the microplate reader (1) in order to measure the fluorescence of the sample.
h) measuring the light emitted from the sample in the wells (3) of the microplate (4) inserted in the microplate reader (1) to measure the luminescence of the sample. The control unit (6, 6 ') comprises a heating device in heat exchange communication with the gas atmosphere in the sample compartment (19), the gas atmosphere in the sample compartment (19) being heated by the heating device The method according to claim 16 , characterized by The control unit (6, 6 ') comprises a temperature sensor in the sample compartment (19) and the temperature of the gas atmosphere in the sample compartment (19) is measured to control the temperature in the sample compartment (19) The method according to claim 16 , characterized in that: Method according to claim 16 , characterized in that the humidity of the gas atmosphere (7) in the sample compartment (19) is controlled using a control unit (6, 6 '). The concentration of the at least one gas is measured using the gas sensor of the control unit (6, 6 ') and the predetermined gas atmosphere (7) is adjusted in the sample compartment (19), said steps f), g), h The method according to claim 16 , characterized in that at least one or more of) are carried out in the regulated gas atmosphere of step d). The reagent is added to at least one sample in at least one well (3) of the microplate (4) using the injection device (10) to obtain at least one measuring device (2 ', 2 ", 2"' 17. A method according to claim 16 , characterized in that the chemical reaction is detectable using 17. A method of measuring a living cell in the microplate reader (1) according to claim 16 , comprising:
A method wherein the living cells are measured in a controlled gas atmosphere (7) and the living cells are selected from the group comprising microaerophilic, facultative anaerobic and obligately anaerobic microorganisms, fungi, eukaryotic cells.

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